Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators.

A ll microorganisms need to rapidly sense environmental changes and to respond to them in order to survive, adapt and reproduce. This response is mainly mediated by transcription factors (TFs) that regulate the expression of relevant proteins by either blocking or favoring the activity of RNA polymerases at specific gene sites 1,2 . Besides their cognate DNAbinding site region, TFs generally have a domain that recognizes effectors, such as other proteins or small ligands. Members of the CRP-FNR and Rrf2 families of dimeric microbial TFs can use the gas-binding affinity, redox properties and intrinsic lability of iron-sulfur clusters to modulate their DNA binding [3][4][5][6][7] . The wellstudied Fumarate Nitrate Reductase regulator (FNR) uses a [4Fe-4S] cluster to sense O 2 levels and to regulate a dimer ↔ monomer transition mediated by cluster disassembly and a metastable protein interface 8 . Only the dimer binds to specific DNA sites 9 . The Rrf2 family was first described in Desulfovibrio vulgaris Hildenborough 10 and the three-dimensional structure of the cysteine metabolic regulator CymR from Bacillus subtilis (Bs), which does not bind an iron-sulfur cluster, was the first to be reported for a member of this family 11 . Structural and functional studies of the related Staphylococcus aureus (Sa) CymR revealed a sensing mechanism in which DNA binding is lost under oxidative stress conditions after oxidation of a surface cysteine thiol 12 . BsCymR binds to DNA after forming a complex with O-acetylthiol-lyase only when cysteine is present 13 .
Members of the Rrf2 family that coordinate iron-sulfur clusters use them to sense various effectors and regulate gene expression to enable adaptation, maintenance of homeostasis and cell protection. Characterized cluster-binding TFs include: IscR from Escherichia coli (Ec) and Thermincola potens (Tp) that sense cell iron-sulfur cluster levels and oxidative stress 6,14 ; RsrR, a redoxsensitive response regulator from the soil bacterium Streptomyces venezuelae (Sv) 5 ; Rhizobium leginosarum (Rl) RirA that uses a labile [4Fe-4S] cluster to regulate cellular iron concentrations 15 ; and NsrR, a regulator of nitric oxide (NO)-induced stress in many bacterial species 4,16 . The regulation of their DNA binding capabilities depends on protein conformational changes caused by the status of the cluster or its absence. The first crystal structures of an iron-sulfur sensor of the Rrf2 family were those of the apo-form of Ec-and Tp-IscR 17,18 . More relevant to the work reported here, the structure of apo-EcIscR in complex with a 29 base pair (bp) fragment of the hydrogenase-1 hyA operator was also solved 17 . The structure of [2Fe-2S]-IscR, which recognizes different operator sequences 19 , has not yet been determined, either alone or in complex with DNA.
We have reported the crystal structures of [2Fe-2S]-SvRsrR alone 20 and in complex with cognate DNA, the latter at medium resolution 21 . Using a combined approach of structural and mass spectrometric studies and molecular dynamics calculations, we were able to show how the one-electron cluster reduction of [2Fe-2S]-SvRsrR triggers protonation of a histidine residue and the burial of a tryptophan side chain, causing conformational changes that lead to DNA dissociation 21 . Our groups have also reported the holo-and apo-crystal structures of NsrR from the antibioticproducing bacterium Streptomyces coelicolor (Sc) 22 . The principal regulatory target of this TF is, in most organisms, the hmp gene, which encodes a flavohemoglobin that converts NO to nitrate (NO 3 − ) or nitrous oxide (N 2 O) under aerobic or anaerobic conditions, respectively 23,24 .
ScNsrR is so far the only NsrR to have been characterized in significant detail 22,[24][25][26][27][28] . Its regulon is small, enabling a detailed investigation of the relative DNA binding properties of [4Fe-4S]-ScNsrR to each identified operator: hmpA1, hmpA2, and nsrR, showing a 23-base pair (bp) consensus recognition site. Tightest binding was observed for hmpA1, followed by nsrR and hmpA2. 25 In the presence of NO, the ScNsrR [4Fe-4S] cluster is rapidly lost, causing the dissociation of the protein-DNA complex. Binding of hmpA2 was found to be the most sensitive to NO, followed by hmpA1 and nsrR 24 . Apo-ScNsrR (or [2Fe-2S]-ScNsrR, which could be generated and stabilized upon O 2 exposure in the presence of β-mercaptoethanol) 29 did not bind DNA with significant affinity 25 . In some bacteria, such as Ec and Salmonella enterica serovar Typhimurium, many more genes are under NsrR control, suggesting a broader regulatory role for NsrR in these microorganisms 30,31 .
A comparison of the three-dimensional structures of holo [4Fe-4S]-ScNsrR and apo-ScNsrR furnished a first model to understand how NO binding to the iron-sulfur cluster, and the resulting conformational changes, control the configuration of the protein DNA-binding surface 22 . Here, we report the 3.0 Å resolution X-ray structure of ScNsrR complexed with a 23-bp hmpA1 operator fragment and use it, alongside the structures referred to above, to establish what determines the DNA binding specificity among the three structurally-related iron-sulfur Rrf2 family proteins IscR, RsrR and NsrR. DNA recognition is generally considered to depend on either a direct readout of the nucleobase sequence, a readout of a sequence-dependent DNA phosphate backbone shape, or a combination thereof 32,33 . Related to the recognition process is the flexibility of the DNA operator regions and their propensity to undergo conformational changes upon protein binding. In a classical study 34 , a crystalline B-DNA dodecamer went from an almost straight double helix structure to a bent one as the temperature was changed from 7°C to 20°C. DNA flexibility has also been shown by solution NMR studies: the 31 P chemical shifts of double stranded (ds) B-DNA fragments vary as a function of the sequence-dependent ratio of B I and B II backbone states. These are in a dynamic conformational equilibrium, with B I being the most commonly observed in regular B-DNA 35 . We have explored these aspects by comparing the remarkably different DNA binding modes of [4Fe-4S]-ScNsrR (this work), apo-IscR 17 and [2Fe-2S]-RsrR 21 in their respective crystal structures. In addition, we have tested four modified oligonucleotides sequences between two recognition sites of the NsrR regulator in order to explore the effect of DNA flexibility on its binding. This analysis allows us to propose a series of structural features that determine specific DNA binding within a class of closely related proteins.

Results
Structure of the [4Fe-4S]-ScNsrR-hmpA1 operator complex. Table 1 shows the statistics of the 3.0 Å resolution structure of [4Fe-4S]-ScNsrR complexed to the 23-bp hmpA1 operator fragment. High atomic temperature (B) factors indicate significant disorder, in agreement with the flexible and dynamic structure previously observed for the noncomplexed protein structure 22 . Both the operator fragment and the bound protein dimer are about 80 Å long (Fig. 1). The buried surface area (BSA) of the protein/DNA complex is 4222 Å 2 , comparable to the BSA of 4981 Å 2 of the NsrR dimer but much larger than the 1996 Å 2 buried between the two strands of the DNA helical fragment. The BSA at the protein/DNA interface of the EcIscR/hyA complex 17 is 3780 Å 2 . This relatively low value is explained by the fact that some of the amino acids of EcIscR facing the central region of the hyA operator were not resolved and therefore not included in the structure. A higher value of 4490 Å 2 is obtained for the SvRsrR/rsrR complex 21 because the larger 39 bp rsrR operator fragment covers the protein sensor more extensively than the 23 bp hmpA1 one does. Residues 59-64 of the ScNsrR dimer wings, which are disordered in the free protein 22 , are partially resolved in the complex where they interact with the ends of the hmpA1 operator fragment (Fig. 1). Other DNA binding regions include the N-termini of α-helices 1 and 2 and the side of the third one, which corresponds to the regulatory helix (RH) of a helix-turnhelix (HTH) motif. Conversely, the C93-C99 turn, which we previously considered as a potential additional contact interface 22 , does not directly interact with the DNA. Compared to many other proteins with a winged (w) HTH motif 36 , the DNA binding region of ScNsrR contains an additional preceding αhelix and, consequently, can be better described as an H-wHTH motif.
Structure of the DNA hmpA1 fragment. The 23 bp hmpA1 operator fragment has a B-DNA conformation. The double helix runs almost straight through the central 11 bp region and bends at its extremes around α-helix 3 of each of the NsrR subunits (Fig. 1). The distance between phosphate P atoms across the minor groove (MiG) decreases from 12.6 Å at the center of the fragment to 9.6 Å between nucleotides 0 and + 4 of the other strand. It increases again to about 13.8 Å between nucleotides +9 and −6 before narrowing to 10.3 Å between nucleotides + 11 and −7. Minimal MiG distances are observed for the + 4 and −7 phosphates, which respectively interact with the His42 and His52 imidazole groups. An analysis of torsion angles with the program DSSR 37 (Fig. S1) shows that 32 of the 44 phosphate groups in the bound 23 bp hmpA1 operator fragment (excluding the 5'-terminal ones) are in the B I conformation. Conversely, the phosphates bound to the C3' atom of the 0 and + 2 nucleotides are in the B II conformation (see also Fig. S2), while the remaining ones are in a conformation intermediate between B I and B II (Fig. 2). The reorientation of the T +2 3'-phosphates from B I to B II conformations causes propeller twists of −19.0°for bp C +3c /G -3d (Figs. 3 and S3) and −15.9°for bp T -3c /A +3d .
Protein-DNA interactions. Most of the ScNsrR contacts are made with the phosphate backbone of the hmpA1 operator (Figs. 2, 3 and Table S1). The His42-Nδ1 and His52-Nε2 atoms interact with the respective 5 ' -phosphate groups of nucleotides +4 and −7 in opposite strands (Figs. 1-3). These interactions could be salt bridges if the Nδ1 and Nε2 atoms of the imidazole groups are protonated. The side chains of Arg2, Lys5 and Lys45, present at the contact interface, are disordered ( Fig. S4), as they were in the 1.95 Å resolution structure of non-complexed [4Fe-4S]-ScNsrR 22 . However, these basic residues could still interact with the negatively charged DNA phosphate backbone (Fig. S5). The positive dipole moments at the N-termini of α-helices 1 and 2 provide additional electrostatic stabilization via H-bonds from: (i) Lys5 main chain N atom to OP1 and Phe6-N to OP2 of the 5 ' -phosphate group of the +3 nucleotide, which is in a B II conformation (the non-complexed ScNsrR structure has a bound sulfate at this position, see Fig. S6); (ii) Thr29 main chain N atom to the 5 'phosphate of the −9 nucleotide in the opposite strand, which is also hydrogen bonded to Thr29-Oγ1. The Thr41 and Thr48 Oγ1 atoms of α-helix 3 make similar H-bonds to the respective 5 'phosphates of the +5 and −8 nucleotides in opposite strands.
There are only a few specific interactions between ScNsrR amino acid side chains and the nucleobases of the hmpA1 operator ( Fig. 3 and Table S1). In the major groove (MaG), Lys45 could establish H-bonds to ring atoms of G −6 and surrounding bases. However, its weak electron density (Fig. S4)   its guanidinium group and the O2 atom of the pyrimidine base at the −9 position and van der Waals (vdW) contacts with T +10 -O2 and G +9d -N2. Gly61 makes vdW contacts with T +11 -O2. Basespecific vdW contacts are observed in the MaG from Thr41 A (4 atoms), His42-Cδ2 and Tyr40 B -Oη to the C7 atoms of T +5d , T +4 and T -9d , respectively (Table S1). Additional resolved interactions with deoxyribose atoms involve the RH Thr41 and Thr48 and the wing Arg60.
Comparison of the protein-DNA complex with DNA-free ScNsrR structures. A cavity analysis indicates that the putative access path for NO that runs along the cluster ligand Asp8, previously observed in [4Fe-4S]-ScNsrR 22 , is also present in its complex with the 23 bp hmpA1 operator fragment (Fig. S7). The most important structural difference in the latter is a rearrangement of the region following α-helix 5 ( Fig. 4a) caused by the electrostatic repulsion between the negatively charged Glu87 and the phosphate backbone. A similar effect could have provoked a small shift of Asp96 away from the DNA (see also Fig. S6). A much larger structural rearrangement of the 87-105 region is observed in the apo-3CA-ScNsrR variant where the cluster-coordinating Cys93, Cys99 and Cys105 were substituted by Ala (Figs. 4b and S8). In this variant 22 , helix 5 is kinked at Gly86 and extends to Gly95. Consequently, the C93-C99 loop, which in the [4Fe-4S]-ScNsrR-DNA complex faces the MiG of the hmpA1 central region (Fig. 4a), adopts an entirely different conformation. Because α-helices 1, 2 and 3 are rearranged in the apo structure, its complex with hmpA1 DNA is not stereochemically possible as it would involve several severe clashes with the N-terminal regions of these helices (Fig. 4b).  also Table S1). Circles depicting phosphate (P) groups are colored according to the corresponding minimal P-to-P distance across the MiG relative to its average value of 11.7 Å in regular B-DNA (ΔPP MiG ). Small circles and black stars indicate phosphate torsion angles in the B I and B II backbone conformation, respectively; asterisks highlight those with an intermediate conformation. Comparison with other sensors of the Rrf2 family. The homologous E. coli NO sensor displays several amino acid sequence differences with ScNsrR in the DNA contacting region (Fig. 5). For example, ScNsrR Thr29, Thr41 and Thr48, which are hydrogenbonded to phosphate groups of hmpA1, are neither conserved in EcNsrR, nor replaced by Ser, which would allow a similar interaction. In addition, the phosphate-binding His52 is substituted by Arg. These dissimilarities could explain why ScNsrR does not display significant affinity for EcNsrR-specific DNA sequences 25 . The above mentioned His and Thr residues are also not conserved in SvRsrR or EcIscR, the other two Rrf2 family iron-sulfur cluster binding sensors with known DNA complex structures, although Thr41 is replaced here by Ser. His42, the other ScNsrR residue that possibly makes a salt bridge with the phosphate backbone, is replaced by Tyr in those sensors. Amino acid sequence comparisons, including RlRirA and Sa and BsCymR, reveal a conserved Lx 3 Gx 6 Gx 2 GGx 2 L motif (Fig. 5) that spans the C-terminal region of helix 3 and the wing region. The comparison shows that most of the ScNsrR DNA contacting residues are not conserved in the eight Rrf2 family members. This indicates, as might be expected, that the different binding modes observed for these sensor/DNA complexes result from a combination of specific amino acid (Fig. 5) and nucleotide (Table 2) differences. A detailed comparison of the ScNsrR/hmpA1 and EcIscR/hyA complex structures reveals a small number of base-specific interactions (Table S1). Modelling different operator sequences on the structures of hmpA1 and hyA produces vdW collisions with the respective ScNsrR and EcIscR protein structures (shown in bold italics in Table 2).
DNA operator fragments conformations. The DNA double helices of the 39-bp rsrR, 29-bp hyA and 23-bp hmpA1 operator fragments display different shapes in their respective complexes to SvRsrR, EcIscR and ScNsrR (Fig. 6). The DNA structures accommodate the different positions and orientations of the H-wHTH motifs found in the bound proteins (Fig. S9). The MiG and MaG P-to-P distances (Fig. 6b, c) and widths (Fig. S10) observed at different locations of the DNA double helix in the three complexes are remarkably different. The MiGs are especially different in the central 7-bp region (Fig. 6b, c) for which no base-specific contacts with the protein are observed ( Fig. 2 and Table S1). These groove width variations condition the distance between equivalent operator regions (Fig. S11), which could guide recognition by different Tfs 32,33 .
To investigate whether the sequence of the central 7-bp region of hmpA1 is important for DNA recognition by ScNsrR we performed EMSA binding studies of fragments with modified bases (Fig. 7), embedding the 23-bp consensus central region in 151-bp dsDNA probes (see Methods). Wild type hmpA1 (Av003) has four dinucleotides in a B II phosphate backbone conformation in the 7-bp central region: C 0c A +1c , T +2c C +3c , G 0d A +1d and T +2d A +3d (Figs. 1-2 and 6). Because XT dinucleotides have a low probability of being in the B II conformation in regular B-DNA 38 , we replaced A +1c and A +1d by T in probe Av004 and C +3c and A +3d by T in Av005 (see Table S2). The observation that some XT steps are found at the corresponding positions in the aligned hyA and rsrR operator fragments (Fig. 6a) suggested to us that they could play a discriminating role in DNA recognition between b Superposition to apo-3CA-ScNsrR (gray). The complex is colored as in Fig. 1 and selected amino acid residues are highlighted. Red arrows indicate movements observed for residues 87, 93 and 99. Regions of the apo structure that would collide with hmpA1 are indicated by red asterisks. Atom color codes are as in Fig. 3. Rotated stereo zooms of the same superpositions are given in Fig. S6 and S8. NsrR, IscR and RsrR. However, our EMSA results did not show any clear difference in binding affinity to ScNsrR between the two constructs having XT steps (Av004 & Av005) and the unmodified hmpA1 (Av003) sequence (Fig. 7a-c). We also checked the binding of variants with an AT-rich (Av006) and a GC-rich (Av007) central region (Fig. 7d-e), because AT-rich base sequences have been associated with an increased likelihood of MiG narrowing 32 , as observed in the central region of the 23-bp hmpA1 fragment (Figs. 2 and 6). However, again our results showed that ScNsrR binds the modified operators essentially stoichiometrically and we could detect no significant differences in binding affinities under these conditions. It should be noted that variations in the flexibility of the central region caused by A/ T or G/C enrichments 39-41 could affect DNA-protein binding kinetics. However, such an effect would not be detected by the EMSA assay reported here.

Discussion
In spite of the large buried complex surface, most of the ScNsrR contacts with the hmpA1 operator are with the phosphate/sugar backbone; only a few direct interactions with nucleobase rings are observed (Figs. 2 and 3). What, then, determines the hmpA1 specificity for ScNsrR? Like most other DNA-binding proteins, sensors such as NsrR must locate specific targets in order to perform their biological role. Experiments performed with green fluorescent protein (GFP)-tagged lac repressor showed it to diffuse along the helical axis of the DNA probe via a sliding mechanism 42 . Furthermore, a similar study using an RNA polymerase demonstrated that this protein tracked the helical pitch along a DNA groove 43 . These interactions, made of non-specific electrostatic contacts of protein residues with the phosphate backbone, are dominated by a fully entropic potential 44 . Indeed, the electrostatic potential at the ScNsrR surface is consistent with such non-specific interactions playing an important role in DNA recognition (Fig. S5). Specific binding will then require a unique DNA sequence and protein-nucleobases interactions conditioned by the shape of both partners 45 .
The shape of B-DNA is determined by sequence-dependent variations in minor and major groove width and curvature that are at least partially coupled. Inter bp rolls, defined as rotations around the base pairing axis perpendicular to the double helix axis, are major contributors to DNA curvature 46,47 ; zero rolls give straight B-DNA double helices 48 . The hyA central 11-bp region is curved, as reflected by an average bp roll of 2.3° (Fig. S1). This value is only 0.5°in hmpA1, in which the same region is almost straight. This may be related to the observed presence of four B II backbone conformations in hmpA1 (Fig. 2). Dinucleotide bps, such as TpC•GpA, with both connecting phosphates in a B I conformation (i.e., B I •B I ) are mostly associated with positive rolls, whereas negative rolls are typically found for B I •B II conformations 49 . As a result, the rolls of a sequence of bps with alternating B I •B II and B I •B I phosphates (like in hmpA1) may partially cancel out, producing a smaller average roll than a sequence lacking B II conformations (like in hyA). However, our EMSA DNA binding studies (Fig. 7) do not suggest any role, at least in vitro, for the observed B II backbone conformations in hmpA1 binding to the protein sensor.
Different backbone torsion angle combinations are required for successive dinucleotides to bend in the same direction along the DNA double helix. Factors such as base orientation and stacking, and electrostatic interactions between phosphate groups may influence the torsion angle values. DNA bending towards the MiG, which narrows it, is energetically more costly than its bending towards the MaG 50 . However, the electrostatic repulsion between MiG opposing phosphate groups is greatly reduced by the proximity of positively charged amino acid sidechains. Thus, a minimal MiG width (MiGW) is observed at the + 4 phosphate of hmpA1 and at the +2 phosphate of hyA, which respectively make salt bridges with ScNsrR His42 (assuming it is positively charged) and EcIscR Arg2 (Fig. 6). Narrow MiGs in naked DNA have been associated with A/T bp-rich sequences 32,46 . Stella and coworkers found that the A/T-rich central region of the Fis protein high-affinity DNA binding sites displayed a small MiGW 46 . G/C substitutions in this region increased the MiGW and resulted in lower DNA binding affinity, an effect these authors explained by the increased conformational cost of compressing the MiG. However, B-DNA sequences are known to form dynamically heterogenous structures that explore a large conformational space 51 . When we performed an EMSA binding study of a variant of hmpA1 with 7 G/C bps in the central region, we did not observe a change in binding affinity to ScNsrR (Fig. 7). Neither did we see an effect when the region was enriched in A/T bps. This may imply that the flexibility of the hmpA1 DNA central region of the structure does not have a marked effect on the thermodynamics of the recognition of the operator -through either induced fit or conformational selection-by the different shapes and electrostatic potentials at the ScNsrR surface. A possibly kinetic effect would not be detected in the EMSA.  In view of these results, direct protein-to-DNA interactions, which mostly involve the RH and the wing (Figs. 2 and 6), are probably much more important for operator recognition. The DNA segments bending around the regulatory helix display a widening of the MaG. This widening is more pronounced in hyA than in hmpA1 and rsrR (Fig. S10), which could be due to the compression of the MiG between the P +2 phosphates of the neighboring central region in hyA (Fig. 6). The wing regions of ScNsrR, EcIscR and SvRsrR, which display the highest amino acid sequence homologies within Rrf2 family proteins (Fig. 5), interact with a more similar, relatively A/T-rich MiG (Figs. 2 and 6a). In hmpA1, as in hyA, the corresponding MiGWs are narrower than in rsrR. This is probably due to the close interaction of the positively charged ScNsrR Arg60, and EcIscR Arg59, with deoxyriboses and base rings at the MiG edge ( Fig. 6c and Table S1). These interactions also compensate for the electrostatic repulsion between opposing phosphate groups. The broader rsrR MiG in the region that binds the protein wing could be explained by its lower A/T bp content and by the fact that in SvRsrR this Arg residue is substituted by Gln57 (Fig. 5).
To further analyze DNA binding specificity, we modeled the hyA and rsrR operator sequences (Fig. 6a) on that of hmpA1 in its complex with ScNsrR. Substituting hmpA1 C +6 by (hyA) T +6 introduces a vdW (d = 2.7 Å) collision between the added C7 methyl group and the Cγ2 methyl of (ScNsrR) Thr41 (Table 2). Because the latter is located in the N-terminal region of the RH and its Oγ1 atom makes a H-bond with the 5 ' -phosphate group of the same base in hmpA1 (Table S1), other interactions involving the RH are likely to be perturbed as well. The same substitution in the other DNA strand should similarly modify the RH of the corresponding ScNsrR subunit, which could be sufficient to abolish binding. Similarly, when modeled on the structure of hyA, the C7 methyl group of T +5 found in one strand of hmpA1 and in both strands of rsrR lies within 2 Å from the side chain of Ser40 of EcIscR (Table 2). In addition, the T -8d C7 methyl group of hmpA1 would collide with the carboxylate group of EcIscR Glu43, which has been assigned an important role in DNA recognition 17 . These collisions suggest that hmpA1 and rsrR are not recognized by EcIscR. Because of its relatively low resolution 21 , we refrained from doing a similar analysis using the structure of the SvRsrR/ rsrR complex.
The observed interaction of Pro61 in the wing of EcIscR with the MiG of a 29 bp fragment of hyA (Table S1) suggests that the aligned Arg62 of ScNsrR could bind to the MiG of a longer than 23 bp fragment of hmpA1, as the one used in our EMSA studies which is 151 bp-long (Fig. 7). This should increase binding affinity. Substitution of (hmpA1) T +10 by (rsrR) G +10 introduces vdW collisions with (ScNsrR) Arg60 in both wing regions ( Table 2). We also noted that the last three Gly residues of the conserved Lx 3 Gx 6 Gx 2 GGx 2 L wing motif directly interact with the DNA in the EcIscR/hyA complex structure (Table S1). This further underscores the importance of the wing region for DNA binding.
There are few structural changes in [4Fe-4S]-ScNsrR upon its binding to the hmpA1 operator (Fig. 4a). As mentioned in the Introduction, the nsrR gene codes for ScNsrR, whereas the two hmpA genes encode NO-detoxifying flavohemoglobins. Full binding of the [4Fe-4S]-ScNsrR dimer to 23 bp DNA stretches -included near the center of 267 bp constructs-as investigated by earlier reported EMSA studies 25 , requires corresponding ratios of 1:1 for hmpA1, 2.5:1 for nsrR and 4:1 for hmpA2. NO-induced structural changes resulting from iron-sulfur cluster modification and degradation will alter the ScNsrR structure and, consequently, modify its binding affinity. The [4Fe-4S]-ScNsrR TF binds to its known operators 25 with much higher affinity than either non-physiological [2Fe-2S]-ScNsrR species 29 or its apoform. We have provided structural data to show that the loss of the iron-sulfur cluster in the latter introduces radical structural differences relative to the holo protein form at the DNAinteracting region 22 . Large conformational changes, including the possible formation of persulfide bonds 27 , should also be present in [2Fe-2S]-ScNsrR; indeed these bonds have been observed in partially oxidized [2Fe-2S] clusters of HydE 52 and detected by resonance Raman spectroscopy in FNR 53 .
We have found that the DNA-free [4Fe-4S]-ScNsrR structure does not change much after binding of the 23 bp hmpA1 operator fragment and that changing the sequence of its central 7-bp region (which shows no direct base-protein contacts within 4 Å) has no effect on the binding affinity. Consequently, it seems reasonable to assume that the DNA backbone conformation of bound hmpA2 and nsrR operators is not very different from that of hmpA1. We have therefore modeled the hmpA2, nsrR and (Ec) hmpA sequences on that of hmpA1, knowing that (Ec) hmpA binds only very weakly to ScNsrR 25 . Analysis of the resulting predicted complex structures shows a number of bad contacts involving Arg60 in the two NsrR wing regions. Together, pointing into the minor groove at the two extremes of the hmpA1 operator fragment, Arg60 A and Arg60 B make close (<4 Å) contacts with eleven nucleotides (Fig. 2 and Table S1). A collision with one of these nucleotides should perturb the interactions with the other ones. Three bad (d < 2.85 Å) vdW contacts with Arg60 are obtained for the modeled ScNsrR/Ec-hmpA complex (Tables S3-S4 and Fig. S12), which could explain why no significant binding is observed for this operator. One bad contact with the Arg60 guanidinium group is obtained for nsrR, in line with its reduced binding affinity. The observed low affinity of hmpA2 is possibly explained by the substitution of T -9d by C and T +5d by G causing a loss of favorable vdW contacts with the T-C7 methyl groups.
Holo-ScNsrR-operator binding is abolished for NO-to-[4Fe-4S]cluster ratios of about 2 for hmpA2, 4 for hmpA1 and 8 for nsrR 24 . As in the case of O 2 oxidation, important conformational changes at the cluster region are expected when intermediate NO-bound degradation products are formed. Kinetic and spectroscopic studies have shown that these intermediates appear at ratios of about 2, 4, and 6 bound NOs per cluster 24 . They share spectral features with known NO-iron complexes such as [Fe(NO) 2 (RS) 2 ] (dinitrosyl iron complex), [Fe 2 (NO) 4 (RS) 2 ] (Roussin's red ester) and [Fe 4 (NO) 7 (S) 3 ] (Roussin's black salt, RBS) 24,26 . In the case of the RBS-like intermediate, it has been suggested that Cys thiolates (or persulfide derivatives of these) replace sulfide ligands 26,28 ; this would explain how these nitrosylated moieties bind to the protein after the loss of cluster sulfide ions. Several NO-modified cluster forms have also been characterized by mass spectrometry, including early mono-and di-nitrosyl intermediates of the [4Fe-4S] cluster 28 22 has defined the nature of the conformational changes induced by either iron-sulfur cluster modification or loss that will preclude sensor binding to hmpA1 (Fig. 4b). Because we have not yet characterized any NO-complex of ScNsrR, we have at this stage no structural data to explain the different NO sensitivities of its complexes with hmpA2, hmpA1 and nsrR.
In conclusion, the main determinant factors for the ScNsrR base sequence specificity can be described as follows: (i) the end regions of the hmpA1 operator interact extensively with the ScNsrR wings (this is also the case for hyA and rsrR, which respectively bind the apo-EcIsrR and [2Fe-2S]-SvRsrR wings); (ii) important structural differences between the bound operators of these three Rrf2 family members reside in variations of minor and major groove widths, which result in different phosphate positions; (iii) the DNA also displays specific curvatures around the RH of the sensor that properly orient its phosphate groups to form salt bridges and hydrogen bonds with equivalent side chains from both protein subunits; (iv) much of the specificity involves a relatively small number of direct interactions of protein side chains with nucleobase rings; (v) as shown in the EMSA experiments, changes in the flexibility of the hmpA1 central region do not affect the thermodynamics of NsrR binding (whether they modify the kinetics of this process was not investigated here). Solvent-mediated interactions may also play a role, but these are not resolved in our 3 Å resolution X-ray model. Our results extend the findings of a recently reported sequence similarity network analysis of the entire Rrf2 superfamily that places ScNsrR, EcNsrR, EcIscR and SvRsrR in distinct cluster configurations 54 . Our next challenges will be to crystallize complexes of [4Fe-4S]-ScNsrR with longer constructs of the hmpA1, hmpA2 and nsrR operators and solve their structures at the highest possible resolution. These structures should help us determine if and how the above-described determinants vary with the nature of the operator. In addition, we aim now at structurally characterizing NO-induced changes for at least one ScNsrR/DNA complex.

Methods
Crystallization. [4Fe-4S]-ScNsrR was purified under strictly anaerobic conditions as described previously 22,25 . A stock solution with a protein concentration of 43 mg/mL in 50 mM Tris pH 8.0, 2 M NaCl, 5% glycerol was used for all experiments. A 23 bp dsDNA fragment of the hmpA1 operator was prepared by annealing its 5 ' -AACACGAATATCATCTACCAATT-3 ' and 5 ' -AATTGGTA-GATGATATTCGTGTT-3 ' complementary oligonucleotides in DNase-free water (Alfa Aesar), as already described previously 24 . [4Fe-4S]-ScNsrR was complexed with the 23 bp hmpA1 operator by incubating a solution of 17 mg/mL of the protein mixed with a 3-fold excess of the dsDNA fragment in 50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol for 60 minutes at 20°C. Crystals were obtained by screening 864 different conditions from commercial kits in 96-well plates with a Gryphon robot (Art Robbins Instruments, CA, USA) installed inside an anaerobic glove box, using the vapor diffusion method. For each crystallization condition a drop was set by the robot by mixing 200 nL of the solution of the [4Fe-4S]-ScNsrR/ hmpA1 complex with 200 nL of the commercial reservoir solution. The sitting drop was then equilibrated against 100 μL of the latter solution. Manual optimization of the best automated crystallization condition yielded usable crystals from hanging drops prepared by mixing 1 μL of the protein-DNA mix with 1 μL of the reservoir solution containing 27% PEG 550 MME, 13.5% PEG 20000, 30 mM MgCl 2 , 30 mM CaCl 2 and 100 mM MOPS/HEPES pH 8.0 equilibrated against 1 mL of this solution. All these experiments were performed anaerobically in a glove box.
X-ray data collection. Crystals were transferred from the original drop to the crystallization solution, with or without 20% v/v glycerol added as cryoprotectant. They were then fished with a cryo-loop, flash-cooled in liquid propane inside the glove box 55 and transferred outside for storage in liquid nitrogen. Initial X-ray diffraction data were collected to 4.1 Å resolution for a crystal mounted from a cryo-protected solution, while maintaining it under a N 2 stream at 100 K at beamline PX-2A of the SOLEIL synchrotron (Saint-Aubain, France). Data to about 3.0 Å resolution were subsequently obtained from crystals flash-cooled without added glycerol. The best data set ( Table 1) was collected at a temperature of 100 K and a wavelength of 1.00001 Å with an Eiger X 9 M (Dectris) detector at beamline X06SA of the Swiss Light Source (Villigen, Switzerland), using the DA+ software 56 . The diffraction data were indexed, integrated and scaled with the XDS package 57 and the program AIMLESS 58 was used to check for anisotropic diffraction. Although X-ray data reduction initially indicated that the crystal belonged to the hexagonal P6 5 22 space group, further analysis showed it to be P6 5 . The change in Laue class from 6/mmm to 6/m of the diffraction data set results from the fact that the dsDNA fragment is not a perfect palindrome; consequently, it does not display the twofold symmetry of the protein dimer. The intensity statistics of the data showed no evidence of twinning.
Structure determination. The structure of the [4Fe-4S]-ScNsrR/hmpA1 complex was solved by molecular replacement (MR) with PHASER 59 using two search models: the known non-complexed protein structure 22 and the 23 bp hmpA1 operator. The latter was constructed with B-DNA geometry using COOT 60 . The obtained MR solutions were confirmed by high peaks in F obs -F calc and anomalous difference electron density maps for the ScNsrR [4Fe-4S] clusters, which were not included in the protein search model. The resulting model was subjected to rigid body, positional, TLS and B-factor refinement with the PHENIX and Refmac5 programs 61,62 , using COOT 60 for manual corrections. Non-crystallographic symmetry and secondary structure restraints were used for positional refinement. H-bond restraints were included for DNA base pairing interactions and some protein-DNA contacts. The refinement procedure gave best results when defining the hmpA1 23 bp operator and two protein half-dimers as TLS groups. No hydrogens were included in the model. Each half-dimer consisted of NsrR residues 1-86 and 124-144 from one subunit and residues 87-123 from the other, including also the [4Fe-4S] cluster bound between them. The final model has good refinement statistics, stereochemistry (Table 1) and a low all-atom clash score of 3.8, as measured by MolProbity 63 . There are 0 outliers in the Ramachandran (φ-ψ) plot and 2.3% rotamer outliers. The dsDNA fragment fits well to the electron density map but a relatively poor match is observed for about 30% of the protein model due to disorder, as indicated by high B-factors. However, all ScNsrR side chains forming hydrogen bonds or salt bridges with the dsDNA have good matching electron density. We tried to explain residual peaks found at both ends of the hmpA1 operator fragment in the final F obs -F calc map by adding additional ordered nucleotides to the model. However, this resulted in an increase of the R free . Because a threefold excess of DNA was used for crystallization, we tentatively assign these residual peaks to partially disordered non-complexed DNA. Other operator sequences were modeled with COOT 60 on the available structures of hmpA1 (this study) and hyA 17 without changing the structure of the DNA backbone.
Structural analyses. Buried surface areas (BSAs) between the various protein and DNA chains were determined with the program Pisa 64 . DNA geometry, including the measurement of torsion angles, bp origins, rolls and propeller twists, was analyzed with DSSR 37 , with groove widths calculated from phosphate-P positions as defined by El Hassan & Calladine 65 (Fig. S10). We also used the in-house program CavEnv (incorporated in the CCP4 package 66 ), which takes into account all atoms of the structure with an atom-type dependent vdW radius, to investigate cavities and groove widths (Fig. S7). Figures were prepared with either COOT 60 , Bobscript 67 and Raster3D 68 , PyMOL (www.pymol.org, the PyMOL molecular graphics system, version 2.1 Schrödinger, LLC) or manually. Compilations of all the used programs were provided by SBGrid 69 . The A/T notation for bps and AT for base steps is used throughout, with a number and one letter for the strand added as an index in some cases. Nucleotides are numbered with respect to the central bp, which is assigned the value zero. Amino acid residues are shown using three-letter codes in the text and one-letter codes in the figures and tables. Structures of the EcIscR/hyA and SvRsrR/rsrR complexes were obtained from deposition codes 4HF1 and 6Y42 in the Protein Data Bank 70 .
EMSA (electrophoretic mobility shift assay) binding studies. DNA fragments carrying wild type or mutated hmpA1 promoter sequences (Table S2) were PCRamplified using a 151-base-pair double-stranded DNA template, 6-carboxyfluorescein (6-FAM) modified primers (Metabion, Germany) and Phusion Hot Start II DNA Polymerase (Thermo Scientific). The PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer ' s instructions. The concentration of the 6-FAM labeled probes was determined using a Varian Cary 50 UV-Vis spectrophotometer. The molecular weights and 260 nm absorption coefficients for 6-FAM labelled probes were calculated using OligoCalc 71 . EMSA reactions (20 μl) were carried out on ice in 10 mM Tris, 54 mM KCl, 0.3% (v/v) glycerol, 1.32 mM glutathione, pH 7.5, as previously described 25 . Briefly, 2 μl of each DNA probe was titrated with varying aliquots of [4Fe-4S] NsrR, to a~20-fold molar excess. Loading dye (2 μl of 0.3% (w/ v) bromophenol blue, 50% (w/v) glycerol) was added and the reaction mixtures were immediately separated at 30 mA for 30 min on a 5% (w/v) polyacrylamide gel in 1 × TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA). Gels were visualized on a Typhoon FLA9000 (GE Healthcare). Polyacrylamide gels were pre-run at 30 mA for 2 min prior to use. Each probe has a dimeric NsrR binding site with one [4Fe-4S] cluster per protein subunit. As apo-NsrR does not bind DNA 25 , concentrations are presented as [4Fe-4S] per DNA, as indicated in Fig. 7.
Statistics and reproducibility. X-ray diffraction data collection and refinement statistics for the reported NsrR/hmpA1 complex are given in Table 1. EMSAs for each tested DNA fragment were carried out at least twice, on different days, to ensure repeatable observations. The images shown in Fig. 7 are the best representative images for each EMSA probe.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Atomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank 70 (https://www.rcsb.org) under accession number 7B0C. A structure validation report is also available there. The two other structures used in our paper are available in the Protein Data Bank (pdb) under accession codes 4HF1 and 6Y42, as given in Fig. 5. These pdb depositions were used to generate all the figures given in the main manuscript, as stated above in the section Structural Analyses, except for the manually prepared Fig. 5 and the measured EMSAs in Fig. 7. For the latter, uncropped images are included in the Supplementary Material as Fig. S13. Interatomic distances in Figs. 1-3, 6b, and c were measured from the atomic coordinates in the pdb depositions given above. Amino acid sequences in Fig. 5 were obtained from the pdb deposition codes mentioned in its last colum or from the UniProt database (https://www. uniprot.org, codes Q1R382 for EcNsrR and Q1ML82 for RlRirA).